i | ICSAFS-UNPAD DISCLAIMER

The International Conference on Sustainable Agriculture and Food Security (ICSAFS) was held on September 2011 at Universitas Padjadjaran. Responsibility for the content of these papers rests solely with the authors. Where trade names are used, this constitutes neither endorsement of nor discrimination against any product by the instituion.

Published by Faculty of Agriculture Universitas Padjadjaran

April 2012

Nurbaity, A., Subroto, E., Setyowati, E.Y., Stanica, F., Bari, I.N., Carsono, N., Mulyani, O., Lehmousloto, P., Teng, P.S., Siswanto, S.Y., Aleksic, S. 2012. Proceeding of International Conference on Sustainable Agriculture and Food Security (ICSAFS). 782p.

Copyright on all papers on the Conference resides with Universitas Padjadjaran Jl. Raya Jatinangor km. 21 Bandung 40600 West Indonesia

ISBN 978-979-8246-11-1 [printed version] ISBN 978-979-8246-12-8 [electronic version]

Technical editing and design: Ichsan Nurul Bari and Gigih Ibnu Prayoga

ii | ICSAFS-UNPAD

FOREWORD

Agriculture as one of leading economic sectors in some countries, is currently facing many problems. This situation could be overcomed by policy and institutional environment which is condusive to increase agricultural productivity while maintaining a sustainable agriculture development and food security. According to this, it is required to develop strategies, a new paradigm, and holistic approach to support the agricultural growth continuum.

In order to make a significant contribution to the better understanding of sustainable agriculture for meeting food security needs and addressing climate change challenges, an International Conference on Sustainable Agriculture and Food Security was held in Bandung Indonesia on 27-28 September 2011. This conference was organized by collaboration of four faculties in Universitas Padjadjaran: Faculty of Agriculture, Faculty of Animal Husbandry, Faculty of Fishery and Marine Science, and Faculty of Agricultural Industrial Technology. Ministry of Agriculture of Republic Indonesia and internationally well-known experts from USA, Finlandia, Singapore, Germany, Malaysia, Romania, Republic of Serbia, China as well as Indonesia were invited as resource speakers.

More than 250 participants from 15 countries attended the conference. The conference shared experiences and views regarding agricultural production in a changing environment towards sustainable agriculture development to maintain food security, and stimulated cooperative research among participating institutions.

About 180 papers are presented and the committee hopes that these papers will be a lsating record of the contributions to this conference and a useful reference for all practitioners in the fields of agriculture in general. Some of the topics presented include critical issues dealing with sustainable agriculture and food security, agrosocio-economy, agritechnology, plant sciences, animal production, and food technology. The committee would like to thank the many reviewers of the papers for their contribution to these proceedings.

The conference and proceeding would have not been accomplished without the support of many individuals, groups and academic units. We owe our gratitude to those who commit and dedicate their self to this conference.

Benny Joy Chair of ICSAFS

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CONTENTS

FOREWORD ...... iii

CONTENTS ...... v

INVITED SPEAKERS ...... 1 New Technologies for The Improvement of Yield and Quality of Beef of Domestic Spotted Breed Aleksi S., M.M. Petoi, V. Pateli, Ž. Noakoi, D. Ostoji, N. Staiši, ad M. Noakoi ...... 3 The Effect of Pollution on Food Security of Floating Net Cage Aquaculture in The Lake Dhahiyat, Y...... 9 Characteristics of Indonesian Lakes and Fisheries Development Lehmusluoto, P...... 20 Opportunities for Sustainable Intensification of Agricultural Practices to Improve Crop Productivity of Small Holding Farmers in West Africa Prasad, P.V. V., Jesse B. Naab, Mamadou Doumbia and Timothy Dalton ...... 30 Management of Water Saving and Organic Based Fertilizers Technology for Remediation and Maintaining The Health of Paddy Soils and to Increase The Sustainability of Rice Productvity in Indonesia Simarmata, T., B. Joy and T. Turmuktini ...... 31 New Fruit Technologies in Europe Stăiă, F...... 48

Epoeig Busiess of Gaut Sheep fo Sall Holde Faes i West Jaa Tawaf, R., D. Heriyadi, A. Anang, M. Sulaeman and R. Hidayat ...... 58 Biotechnology to Ensure Food Security Teng, P.S...... 64 Diversification of Food Products to Support Food Security: Development of Food Products Based on Sorghum Rice and Flour Tjahjadi, C...... 65 Application of Genomics Approaches to Unravel The Functional Biodiversity of Farm Animals Wimmers, K. and S. Ponsuksili ...... 73 Ecological Rain-Fed Agriculture in Semiarid Loess Plateau of Northwest China Xiong, Y...... 74 Biodiversity and Variety Improvement of Crop Plant Zain, S.M...... 75

v | ICSAFS-UNPAD SUSTAINABLE AGRICULTURE ...... 91 Indicators of Agricultural Sustainability at the Regional Level a Case Study of Vidarbha Borkar, P...... 93 Effect of Mulch, Clay and Organic Matter on Soil Chemical and Biological Properties of Sandy Soil and Growth of Physic Nut (Jatropha curcas L.) Djajadi ...... 107 Sustainability of Food Resources by Eco-Farming Implementation: The Role of Farmers Socio Economics Frimawaty, E., A. Basukriadi, J. A.Syamsu, and T.E.B. Soesilo ...... 115 Utilization of Yard to Increase Household Income and Food Security Herliana, S. and Yogi ...... 122 Harmonizing Agriculture, Forests and Fishery Management in the Design of REDD+ in Small Islands of Kepulauan Aru Regency, Maluku Province Mardiatmoko, G...... 135 Farmers are sacrificing their health for production of vegetables Muktamar, Z., S. Sudjatmiko, B. Toha, and M. Asteria...... 141 Phosphorus Recovery from Agroindustrial Wastewater through Struvite Crystallisation: Principles and Applications Muryanto, S., A.P. Bayuseno, E. Supriyo, and B. Hermanu ...... 150 Arbuscular Mycorrhizal Fungi Induced the Content of Isoflavonoid that Reduced Potato Cyst Nematode on Roots of Potato Plants Nurbaity, A, T. Sunarto, M.A. Solihin, and R. Hindersah ...... 160 Analysis of Sustainability of Capture Fisheries Resources Management (A Case in Pangandaran the District of Ciamis West Java) Nurhayati, A., Rusidi, M.H. Karmana, & B. Koswara ...... 166 Remediating The Degraded Land Due to Mining of Pumice Stone in The Northern Part of Lombok Island by Applying Silicate Rock-Organic Fertilizer Priyono, J., C. Sukorahardjo, and A. A. Rahmianna ...... 179

Ealuatio of Etesio Wokes Attitude toad Itegated Faig Sste i Indonesia Putra, R. A. R. S, J. Udomsade, and S. Niyamangkoon ...... 186 Numerous Factors Influencing Food Availability During Harvesting and Lean Seasons in West Timor Suek, J. and H.J.D. Lalel ...... 196 The Characterization and Evaluation of Local Upland Rice Cultivars to Blast Disease (Pyricularia oryzae) Resistance in Southeast Taufik, M., T. Wijayanto, and A. Wahab ...... 204 vi | ICSAFS-UNPAD

Effects of Nitrification Inhibitors on Denitrification in Soils Tindaon, F. and J.C.G. Ottow...... 212 Yields Increasing of Sweet Potato (Ipomea batatas l.), Variety of Beauregard by Organic Cropping System at Desa Cilembu, Sumedang Wagiono ...... 228

AGROSOCIO-ECONOMY ...... 235 The Potentials and Pitfalls of Ecotourism Development on Natural Resources Conservation Area in Indonesia Avenzora, R., and T. Sunarminto ...... 237 Instability in Selected Malaysian Crop Production in Reference to the National Agricultural Policies Borkotoky, P., I. AbdLatif, Z.A. Mohamed and M.N. Shamsudin ...... 247 Public Perception of Food Alternatives for Rice in Bandung Deliana, Y...... 253 Physiological Response of Tomato (Lycopersicon esculentum L.) to Boron Fertilizers Under the Varied Soil Lime Content Karaman, M.R., S. Şahi, N. Geoloğlu, M. Turan, and M. Sadıkoğlu ...... 260 Evaluation of Food Safety Concept in Indonesian Food Security Policies Karmana, M.H., E. Wulandari and D. Supyandi ...... 269 Socio-economic Interfaces of African Indigenous Vegetables in a Subsistence Economy and the Implication for Food Security in Western Kenya Langat, B.K., V.K. Ngéno, V. Mugalavai, L.G. Linnet and S. Yaninek ...... 279

Pootig Foest ad No Tie Foest Cultiatio to Iease Faes Ioe on Small Scale Private Forest (A case study at Tanjung Raya Village, Samarang Sub District, Garut, West Java) Suharti, S...... 287 Environmental Friendly Attitudes of Women in Forest Management Based on Communities Empowerment (PHBM) Sulaeman, M.M. and S. Homzah ...... 297

AGRITECHNOLOGY PLANT SCIENCES ...... 306 Antagonistic Activity of Rhodotorula spp. Against Spoilage-Causing Moulds on Tomatoes Anggita, R.H., A. Oetari, A. Salamah, and W. Sjamsuridzal ...... 308 Indonesian Rainfall Patterns: A Dramatic Shift Awaluddin, M.Y. and J. Kaempf ...... 313 Agronomic Trait Evaluation of Transgenic Rice Line With Db1 Transgene

vii | ICSAFS-UNPAD Carsono, N., N.Fitriani, D. Dono, A. Wahyudin, D. Damayanti, M. Herman, Murdaningsih H.K., and K. Toriyama ...... 320 Residual Effects of Vesicular Arbuscular Mycorrhiza and Bokashi on Growth and Yield of Cilembu Sweet Potato (Ipomoea batatas (L.) Lamb.) Djasmara, S., A. W. Irwan, A. Wahyudin, and Nuryani ...... 325 Reformation of Shifting Cultivation Farming System Towards Permanent and Sustainable Cultivation Herman and S. Suharti ...... 331 The Abilities of Endophytic Fungi from Tomato Roots in Suppressing Root Knot Nematodes (Meloidogyne spp.) in Tomato Istifadah, N., Nurholis and T. Sunarto ...... 339 Non-Irrigated Upland Cultivation - Utilizing The Concept of Transpiration Coeffisient Kramadibrata, A.M...... 345 Prospect and Challenge of The Usage of Portable Near-Infrared Spectrometer to Assess Fruit and Vegetable Quality in Indonesia Kusumiyati, S. Kawasaki and H. Kazunori ...... 356 Enzymatic Production of Monoglyceride Through Esterification System Luna, P., N. Andarwulan and T. Haryati ...... 362 Evaluation Drought Tolerance Level of Sweet Potato (Ipomoea batatas L.) Germplasm from NTT Province Mau, Y.S...... 370 Laboratory Bioassay of Entomophatogenic Fungi Beauveria Bassiana and Metarrhizium Anisopliae for Control of Sweet Potato Weevil (Cylas formicarius Fab.) Mau, Y.S...... 377 Simulation Model For Corn (Zea mays, L.) Planting Time Determination In Dryland Of Timor, East Nusa Tenggara Province Mella, W.I.I., T. Vincentius, R. Pollo, A.S.J Adutae, M.M.J. Kapa, M. Kasim, K. Rantelobo, A. Kedang, and A. Geru ...... 385 Efficacy of New Formulation of 1-Methylcyclopropene for Improving Postharvest Quality of Pelargonium Flower Mubarok, S., M. Serek, and V. Mussmann ...... 395 Genetic Diversity of Morphological Responses ond The Relationships Among Javanese Winged Bean (Psophocarpus tetragonolobus L. DC.) Accessions Nusifera, S., M.H. Karmana, M. Rachmadi, and A. Karuniawan ...... 401 Influence of Fermentation by Using Bacillus licheniformis and Bacillus megaterium on Crude Fiber, Fat, Tannin, and Protein Content of Saba Banana (Musa balbisiana colla) Peel Safitri, R., N.A. Fauzana, and E. Kardia ...... 411 viii | ICSAFS-UNPAD

The Effect of Cymbopogon nardus Linne Rendle on Rice Storage Pest Sitophilus oryzae Linn (Coleoptera: Curculionidae) Sanjaya, Y., M. Halimah and Y.S. Mulyati ...... 415 Feasibility Test of The Biopore Absorption Hole to Improve Infiltration Sistanto, B. A...... 418 Relationship between Slope and Soil Physical Properties A Case Study at Pasirwangi, Garut, Indonesia Siswanto, S. Y., Sandrawati, A., and Sangjaya, M.I...... 427 The Potential of Trichoderma Isolated from Cocoa to Control Black Pod Diseases on Cocoa Pod Sriwati, R., Marlina and Mufakir ...... 432 The Effect of Phosphates Solubilizing Bacteria to The Growth and Crop Production of Corn Plant (Zea mays L.) Surtiningsih, T., D. Puspitasari, and A. Supriyanto ...... 438 Inducing Somatic Embryos of Soybean Glycine max and Glycine soja on Sucrose Concentrations Variation Wahyurini, E...... 445

ANIMAL PRODUCTION ...... 453 Isolation and Characteristic of Lactobacillus sp. Isolated from Milks of Cattle, Goat ad Hoeade Yoguts fo Potetial as Poioti Alias, R., R. Ragupathy, K. Anbalagan, N.W.I. Suhaimy, E.S. Idrus, H. Subramaniam, A.N. Awang, P. Rajandara,and F. Riza...... 455 Live Weight Changes of Cattle as Draft Animal Under The Integration of Oil Palm-Cattle System in Bengkulu Dwatmadji and T. Suteky ...... 460 Characterization of Cellulose Enzyme from Milkfish (Chanos chanos) Gastrointestinal As Potential Agent to Degrade Cellulose Hidayanti A.K., Annisa N.L, R. Erdiana1, Winda A.P, An. Ridhowati, Fikri, B.M. , Miranti D.S, Abrory A.C, Trijoko, and Y.A. Purwestri ...... 464 Layer Productivity as Affected by Different Feeding Portion Indreswari, R., U. Atmomarsono, and H. I. Wahyuni ...... 470 The Effect of Kombucha Supplementation in The Ration on Quails Body Weight and Dressed Carcass Weight Lengkey, H. A.W., E. Sudjana, and T. Widjastuti ...... 478 Occurrence of Pork Derivative in Confectionery Product Upon Malaysia Market Noor Asiah Binti Hassan and Rozila Binti Alias ...... 482

Nutitioal Cotets of Gekos Flesh Gekko spp)

ix | ICSAFS-UNPAD Prastiwi, A., D. Yudhabuntara, W. S. Nugroho, and D.A. Widiasih ...... 489 Effect of PUFA Supplementation on Cholesterol, Fat Content, Water Content, and Protein Content of The Simental-Ongole Cross Bred Meat Riyanto, J. , S. D. Widyawati, and W. Pratitis ...... 495 Anticancer Activity of Chitosan from Local Chitin Waste of Fishery Products In Vitro Rochima, E., and A. Diantini ...... 502 Relationship Between Body Part Measurement, Body Weight and Flying Speed of Racing Pigeon (Columba linia) at Local Tournament of Sprint Racing (Case at Local Tournament of the Sprint Pigeon in the District in of Bandung) Sri Bandiati K.P., D. Garnida, and M. Yusuf ...... 511 Performance of Sheep and Goat with Rotational Grazing Under Oil Palm Plantation Based on Animal Unit Equivalent (AUE) Suteky, T. and Dwatmadji ...... 518

FOOD TECHNOLOGY ...... 525 Optimization of Fish Gelatin Extraction from Starry Triggerfish (Abalistes stellaris) Skin Amin, A.M. and N.H. Alias ...... 527 Optimization of Gelatin Extraction Parameters from Cobia (Rachycentron canadum) Skin Amin, A.M., N. Ibrahim, N.J. Mohamad, and W. M. Wan Maizatul Shima ...... 535 Identification of Pork Contamination in Meatballs of Local Market Using PCR-RFLP Analysis Erwanto, Y., M. Z. Abidin, and D. N. Haryati ...... 544

Carotenoid, Total Phenolic Content, and Antioxidant Activities of Jita Leaes (Plectranthus amboinicus L. Spreng.) Lestario, L.N., L. Agustina, and S. Hartini ...... 551 Optimization Formulation of Functional Beverages Based on Medium Chain Triglyceride (MCT) and Virgin Coconut Oil (VCO) Luna, P., S. Usmiati, and A.N. Alamsyah ...... 560 Chemicals and Appearance Characteristics of Noodles Producing from Composite Flour based on Yam (Dioscorea alata L.) and The Beans Markus , J. E. R, and S. S. Oematan ...... 570 The Effect of Type of Packaging and Storage Time on The Quality of Patchouly Oil Nurjanah, S., S. Zain, T. Pujianto, and A.K. Amaliah ...... 577 The Potency of Banana Tissue Culture Development to Answer the Malnutrition Problems Omar, A., V. Narita, Djajanegara, I.R, Supriadi, Y, and Noriko, Nita ...... 585 x | ICSAFS-UNPAD

Quality Degradation of Mashed Red Chilli Based on Capsaicin During Processing Renate, D., F. Pratama, K.Yuliati, and G. Priyanto ...... 593 Improving the Quality of Meat from Old Cattle through Extended Cooking Setyowati, E.Y. and U. Santosa ...... 599 Increasing of Conjugated Linoleic Acid of Dairy Milk with Additional Rice Meal Fermented and Soybean Oil Suhartati, F.M. and W. Suryapratama ...... 606 Chitin Oligomer Production with Unique Chitinase Bacillus sp Strain SW71 Enzyme from Dams Water Shrimp Wahyuni, S. and M. T. Suhartono ...... 612 LIST OF PARTICIPATING INSTITUTIONS ...... 621 THE BEST OF ORAL PRESENTERS ...... 624 THE BEST OF POSTER PRESENTERS ...... 624 LIST OF SPONSORS ...... 625

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INVITED SPEAKERS

1 | ICSAFS-UNPAD Chitin Oligomer Production with Unique Chitinase Bacillus sp Strain SW71 Enzyme from Dams Water Shrimp

Wahyuni, S.1 and M. T. Suhartono2 1 Chemistry Education, , 2Research Center for Biotechnology, Bogor Agricultural University

Abstract

A chitinase-producing bacterium, Bacillus sp strain SW71 was isolated from Pinrang shrimp dams water, South Sulawesi, Indonesia. The bacteria was grown in the liquid medium supplemented with colloidal chitin (0.5%) as a carbon source for 46 hrs. at 37oC to produce chitinase. The optimal temperature and pH for the chitinase activity were 60-70oC and 7-8, respectively. The chitinase was very stable at 60-70oC up to 4 hrs. The enzyme of 0.0085 IU/mg chitin on 1 and 0,5 % colloidal chitin substrate were used to produce chitin oligomers through incubation at one until twelve hours. The reaction products were analyzed and fractionated using HPLC. Free cell supernatant and purified enzyme were shown tetramer and pentamer composition after incubation during twelve hours.

Keywords: chitinase, chitine oligomer, olisaccharide

Introduction

Chitin is among the most abundant biomass present on Earth. Chitin, a polymer of 1,4-N- acetylglucosamine (GlcNAc) units is the second most abundant polymer in nature, after cellulose. Chitin is present in insect, crustaceans and in most fungi. Commercial chitin can be produced from crabs and shrimp shells. Chitin and its derivatives are of interest because they have various biological activities, such as those of an immunoadjuvant and a flocculant of waste water sludge, and agrochemical uses (Flach et al., 1992). Chitinase plays an important role in the decomposition of chitin and potentially in the utilization of chitin as a renewable resource. Chitin and chitinases (EC 3.2.1.14) have an immense potential. Chitinolytic enzymes have wide-ranging applications such as preparation of pharmaceutically important chitooligosaccharides and N-acetyl D:-glucosamine, preparation of single-cell protein, isolation of protoplasts from fungi and yeast, control of pathogenic fungi, treatment of chitinous waste, and control of malaria transmission (Dahiya et al. 2005). Chitin oligosaccharides are prepared by partial hydrolysis of chitin with hydrochloric acid or enzymatically by degradation and transglycosylation. This preteated chitin is then mixed with a chitinolytic enzyme to hydrolyze it to monomer or oligomer of N- acetylglucosamine. Chitin oligosaccharides or chitin oligomers produced by enzymatic hydrolysis of chitin can be used in human health care. Muzarelli (2010) reported that citin and chitosan as immunoadjuvants and non allergenic drug carriers.Chito-oligomers produced by enzymatic hydrolysis of chitin have been of interest in recent years due to their broad applications in medical, agricultural, and industrial applications, including antibacterial, antifungal, hypocholesterolemic, and antihypertensive activity, and as a food quality enhancer. Chitin and chitosan-derivatives have gained wide attention as effective biosorbents due to low cost and high contents of amino and hydroxyl functional groups which show significant adsorption potential for the removal of various aquatic pollutants (Bhatnagar and Sillanpaa (2009).

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The oeial iteest i the utilizatio of hiti ad its deiaties has led to the eed for inexpensive, reliable sources of active and stable chitinase preparation. Production of inexpensive chitinolytic enzyme is also important in the utilization of shelfish waste to solve environmental problems as well as to promote the economic value of marine product (Wang et al., 1985 ).

Chitinases, a group of enzymes capable of degrading chitin directly to low-molecular-weight product, have been shown to be produced by a number of microorganism. Almost all of the reported chitinase-producing strains will use chitin or colloidal chitin as a carbon source (Wang et al., 1995 ). Some of the chitinases produced by bacterial systems that have gained worldwide research interest for their diverse properties and potential industrial uses.

Material And Methods

Chemicals

Colloidal chitin was prepared from commercial chitin by the method of Arnold and Solomon, (1986). All chemicals were purchased from commercial suppliers.

Microorganism

Bacillus sp strain SW71 was isolated from Pinrang shrimp dams water, South Sulawesi, o Indonesia and maintained at 37 C on Sakai medium agar plate containing: 0.7% (NH4)2SO4, 0.1% K2HPO4, 0.1% NaCl, 0.01% MgSO4.7H2O, 0.05% yeast extract, 0.1% bacto tryptone, 2% colloidal chitin and 1.5% bacto agar).

Enzyme production

The activities of chitinase and protein concentration in the culture supernatant at different stages of growth of Bacillus sp. SW71 were observed to obtain maximal production of the enzyme. Bacillus sp SW71 was cultivated in 10 ml of liquid medium in 50 ml erlenmeyer flask containing: 0.7% (NH4)2SO4, 0.1% K2HPO4, 0.1% NaCl, 0.01% MgSO4.7H2O, 0.05% yeast extract, 0.1% bacto trypton, 0.5% colloidal chitin, pH 7 for 12-20 hours. Ten milliliters of the seed culture was transferred into 90 ml of the same medium and grown for 72 hours at 55oC at water bath shaker (120 rpm). The culture broth was centrifuged for 15 minutes at 8000 g (4oC) and the supernatant was used for enzyme purification.

Purification of Chitinase

Ammonium sulphate was added to the supernatant at 50% saturation. The solution was left overnight at 4oC. After centrifugation for 20 minute at 9000 g (4oC), the precipitate was collected to obtain crude chitinase. The crude chitinase was then partially purified by precipitation with ammonium sulphate, Partially purified chitinase was loaded into chromatography on Butyl Sepharose column (1 x 14 cm) continued by Sephadeks G 100 column (1 x 14 cm) equilibrated with the 50 mM Tris-Cl buffer. The contaminant and unabsorbed protein was washed from the column with the same buffer, and the enzyme was eluted with a linear gradient of 0-30% Tris-Cl buffer pH 8. The active fractions were pooled and concentrated through freeze dry.

613 | ICSAFS-UNPAD Enzyme assays

Chitinase activities were measured with colloidal chitin as a substrate. The reaction mixture consisting of 150 l of enzyme solution, 150 l of buffer and 300 l of 0.5% (db) colloidal chitin was incubated at 60oC, except its stated. The mixture was then centrifuged immediately at 10000 rpm for 4 minutes. The control was prepared by addition of the enzyme to the supernatant obtained after centrifugation of the mixture of substrate and buffer. The amount of reducing sugar produced was measured by the modified Schales method (Imoto and Yagashita, 1971), with GlcNAc as the standard. One unit of activity was defined as the amount of enzyme which liberated 1 mol of GlcNAc per minute.

Hydrolysis with a crude, Amonium sulphate precipitate dan pure enzyme complex. The samples of low molecular weight chitin were prepared by hydrolysis of 1% w/v solution of colloidal chitin in 0,2 M sodium acetate at 60oC for 1 until 12 hours by adding culture supernatant (FC and FCSH), ammonium sulphate precipitate (AS) or pure enzyme (PE) as 0,0085 IU/mg chitin. After that the hidrolysate was heated for 15 min a boiling water bath and then centrifuged.

Results And Discussion

Characteristics of Chitinase-producing strain

A DNA fragment amplified from the genomic DNA of strain SW71 with a set of primers of 16S rRNA was directly sequenced. The 1063 bp were sequenced. The sequence homology was analyzed by BLAST programs. The phylogenetic tree was performed with TreeCon program. The similarity rank analysis showed that the isolat SW71 was closely related to Bacillus species. Bacillus sp. B-3 reported by Karita et al., (2001) is the nearest neighbor (98 homology).

Crude Chitinase Characteristics

Effect of temperature and pH on activity of purified Chitinase

The highest activity of purified chitinase was detected at 60-65oC (Figure 1a) and at pH of7 of Tris-Cl buffer (Figure 1b).

400 140

350 120

300 100 250 80 200 60 150 100 40 Relative Activity(%) Relative Relative Activity (%) Activity Relative 20 50 0 0 40 50 60 70 80 90 4 5 6 7 8 9 10 (a) (b) Temperature (oC) pH Figure 1. Effect of temperature (a) and pH (b) on activity of purified Chitinase

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Effect of temperature on enzyme activity was analyzed by assay the enzyme at various temperatures. The chitinase activities were measured at various pH adjusted by the following buffer: citrate buffer (pH 4-6), phosphate buffer (6-8) and Tris-Cl buffer (8-9), boric acid-borax buffer (pH 9) and glycine-NaOH buffer (pH 10). The optimum temperature of the crude chitinase was 60oC and the enzyme work better alkaline condition. Thermostability of the enzyme was measured by incubation at 60oC, 70oC and 80oC for several hours. The enzyme from Bacillus sp. strain SW71 was highly stable at both temperature of 60oC and 70oC.

The effect of various cations addition the crude chitinase activity was also studied (Figure 2). It was carried out by preincubating the enzyme with each addition in 50 mM of phosphate buffer (pH 8) for 5 minute at 55oC and then their activities were measured at o 60 C. The crude chitinase activity was not inhibited by addition some of metals cation. Ca+2,Ni+2, Mg+2, Co+2 and Li+2, except Mn+2 and Fe+2 appearance have a litte effect to decrease enzyme activity. In case of addition some metal ion had no effect to their activity, this is charasteristics for non metaloenzyme.

120 100

80 2mM 60 5mM 10mM 40

(%) activity Relative 20

0 control Li Fe Mn Zn Mg Ca Ni Na EDTA

Metal ion Figure 2. Effect of metals ion on the activity of chitinase

Thermostability of purified enzyme

Thermostability of the enzyme was determined by incubation at 60, 70, and 80oC for several hours and then measured the residual activity (using 50 mM Tris-Cl buffer, pH 8) at the different temperature. The purified chitinase was highly stable at 60 and 70oC (Figure 3).

120.

100

80 e 60 60 70 40 80

Relative activity (%) activity Relative 20

0 1 2 3 4 Time course of heating (hr) Figure 3. Thermo stability of purified chitinase at various temperature

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1 3

80 6 70 9 60 12 50 24 40 30 20 10

N Acetyl Glucosamine (ug/mL) Glucosamine Acetyl N 0 FCS FCLH AS PE Chitin colloidal

Hydrolyzate

Figure 4. N-Acetyl clucosamine production from chitin oligomer hidrolysate for 1(one) until 24 hours ezymatic reaction (FCH= free cells supernatant, FCLH= heated free cells supernatant on 60oC,AS=Ammonium sulphate precipitate, PE= purified enzyme)

A chitinase purified from culture filtrates of Bacillus sp SW71 efficiently catalyzed a hidrolisys reaction on 1% colloidal chitin substrate in a buffer medium containing ammonium phosphate, converting the with 0,0085 IU/mg chitin had yielded pentamer unit for reaction during 12 hours polymer into N-acetyl glucosamine monomer showed AS and FCLH hydrolysate were most efficient to Hydrolysis chitin colloidal substrate than the others enzyme hydrolysate for 1-9 hours. It is appropriate to use a 1% chitin solutions for hydrolysis and this produces maximal yied N-acetyl glucosamine monomer. HPLC analysis used to detection mono-hexamer chitin olygomer composition on 12 hours reaction time (Table 1). That results showed AS and PE hydrolysates has higher pentamer than dimer and monomer N–acetyl glucosamine concentrations. In this case, the addition of time course to the reaction system resulted in a remarkable increase of the pentamer productions, which are desirable as biologically active oligosaccharides.

Table 1. Composition and oligomer Chitin concentration from HPLC analysis

Reaction Monomer Dimer Trimer Tetramer Pentamer Hexamer Sample time (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (hr) AS 12 - 59,2 - - 197,7 - FCSH 12 73,8 - 60,5 - - - PE 12 88,4 61,4 - - 144,7 -

The optimum temperature of crude enzyme was about 60-65oC. It was similar to that reported by other researchers (Ueda and Arai, 1992, Ueda et al., 1995). The thermo stability of chitinase from Bacillus sp. SW71 was quite remarkable, it is stable at 60-70oC and maintains of its stability even after 5 hours at 60oC in case of crude enzyme. A similar phenomenon was found in case of purified enzyme. This is the first report on bacterial chitinase from shrimp dams that was stable up to for 5 hrs at 70oC.

Nam-soo and Kim (1991) stated that the increased of enzyme thermostability can be explained in two mechanisms, namely intrinsic and extrinsic stabilizing mechanism. In case

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of intrinsic stabilizing mechanism, the molecular structure of enzyme itself is of central important for improved thermal stability. Factors contributing to intrinsic mechanism are electrostatics interaction (e.g., formation of salt bridge), hydrogen bond, hydrophobic interaction, content of aliphatic amino acids, intra molecular disulfide linkage, and compact packing. In case of extrinsic stabilizing mechanism, multipoint interaction of enzyme with other cellular component and the presence of specific thermo-stabilizing factors are important for increased thermostability. Factors contributing to extrinsic stabilizing mechanism are protein-protein contact, bindings of substrates and other cellular component of low molecular weight, prosthetic groups, metal cations etc.

The optimum pH for the chitinase produced by strain Bacillus sp SW71 was nearly neutral or slightly alkaline. These were similar to these of chitinase from Pseudomonas aeruginosa K-187 (Wang and Chang et al., 1997). Other chitinase produced by bacteria belonging to the genus of Bacillus have been reported by several researchers. The chitinase from Bacillus circulans WL-12 and Bacillus strain MH-1 work better at acidic conditon (Watanabe et al., 1992, Sakai et al., 1998). The chitinase from Aeromonas Sp No. 10S-24 was also optimum at an acidic condition (Ueda and Arai, 1992). Chitinase from Beauveria bassiana was reported to be active at pH 9.2, however, it was not thermostable (Suresh and Chandrasekaran, 1999).

The inhibition pattern of metal ions and organic compound on chitinase from Bacillus sp SW71 differ from that of the thermostable chitinase reported by other researchers.

Addition of CaCl2 did not have significant effect on the chitinase activity from Aeromonas so No. 10S-24 (Ueda and Arai, 1992) or Pseudomonas aeruginosa K-187 (Wang and Chang, 1997). However, chitinase from Bacillus sp SW71. was not inactivated by Ca+2. Mostly, thermostable enzymes required metal cation such as Ca+2 as specific stabilizing factors. For examples, a thermostable protease (thermolysin) is stable when Ca+2 ion is present (Nam- Soo and Kim, 1991).

To choose which one enzyme hydrolysate can produce high oligomer, We decide to choosed hydrolysate could produced lower N- Acetyl glucosamine more than 9 hours. So to obtain the chitin olygomer composition, HPLC analysis used to detection mono-hexamer chitin olygomer composition on 12 hours reaction time (Table 1). That results showed AS and PE hydrolysates has higher pentamer than dimer and monomer N–acetyl glucosamine concentrations. In this case, the addition of time course to the reaction system resulted in a remarkable increase of the pentamer productions, which are desirable as biologically active oligosaccharides.

Molecular weight of chitin hidrolysis product very important, because of molecular weight product is dependent to functional characterystic and the best bioactive is oligomer composition (Curroto and Aros 1993, Suzuki 1996, Kolodziejska et al. 2000). Ilyina et al. (2000) reported chitinase from Streptomyces cursanovii which yielded oligosaccharides with degrees of polymerization (DP) ranging from 2 to 9. Due to hydrolitic N-deacetylation, conventional partial acid hydrolysis of chitin gives only low yields of the desired pentamers, hexamers and heptamers (Roby et al.(1987)). Murao et al. (1992) reported a novel chitinase from Vibrio alginolyticus which yielded chitotriose and chitopentaose from colloidal chitin substrate.Yoon (2005) reported that diacetyl-chitobiose (GlcNAc)2 was produced exclusively using a Vibrio furnissii chitinase (chi E, 89 kDa) hydrolysis of chitin treated with dimethylsulfoxide containing lithium chloride. This method represents a novel way for the preparation of colloidal chitin. The preparation of a homogeneous series of chitin/chitosan oligomers with the same distribution of degrees of polymerization (DP) ranging from 2 to

617 | ICSAFS-UNPAD 12, but with various average degrees of N-acetylation (DA) from 0 to 90% is described by Trombotto et al.(2008). In this case, the relative proportion of each chito-oligomer present in the mixture can be estimated precisely as a function of DA considering oligomers of same DP. A chitinase from Tricoderma reesei also showed efficient transglycosylation reaction with the tetramer, giving the hexamer and dimer as the major products (Usui et al. (1990). In our research, chitinase from Bacillus sp. SW71 had enzyme activity 0,121 IU/mL or 0,0085 IU/mg chitin could react with 1% chitin colloidal for 12 hours, was produced chitin oligosaccharides with degrees of polymerization (DP) ranging from 2 to 5.

Degrees of polymerization (DP) chitooligosaccarides very important to biological activity, for examples chitohexaose and chitoheptaose show anti-tumor activity and are efficient elicitors of chitinase activity in melon plants (Roby et al.(1987). Chitooligosaccharides had activity as antibacterial, antitumor, and immunostimulant (Meidina (2005), Semenuk et al. (2001), Suzuki (1996)). Yeon (2004) reported that N-acetyl chitohexaose and chitohexaose had growth inhabited to Meth A-solid tumor cells. Hertriyani (2005) reportet also that chitin oligomers produced from chitinase enzyme from Bacillus licheniformis MB2 could activated in vitro lymphocite proliferation. Tanigawa et al., (1991) reported that no tocixity from chitin oligomers concentration at 400 µg/mL to L929 macrophage cells line, if compared with lipopolysccaride (LPS) tocixity at 100 µg/mL. Koide (1998) reported that Hexa-N- acetylchitohexaose dan chitohexaose as antiallogenic mouse tumor sarcoma. Wahyuni (2006) reported that chitosan oligomers produced from chitosanase enzyme from Bacillus licheniformis MB2 could activated in vitro lymphocite cells proliferation and inhibited some cancer cells proliferation better than chitosan polymer.

Bacause of the fact that chitin oligosaccharides with degrees of polymerization (DP) ranging from 2 to 5 could production from chitinase from Bacillus sp. SW71 with 0,0085 IU/mg chitin had yielded pentamer unit for reaction during 12 hours, the possibility of the preparation by varying condition of hydrolysis is needed of this approach. The result obtained show a clear ooportunity of using the chitinolytic enzyme from Bacillus sp. SW71 for the preparation of chitin oligosaccharides.

Conclusions

Bacillus sp. SW71 isolated from Pinrang shrimp dams water, South Sulawesi, Indonesia, produced thermostable chitinase.The optimum temperature and pH for the enzyme were 60 -79oC and 7 -8, respectively. The enzyme was also stable at 60-70oC up to 4 hrs. The crude chitinase activity was not inhibited by addition of 0,5-10 mM some of metals cation. Ca+2,Ni+2, Mg+2, Co+2 and Li+2, except Mn+2 and Fe+2 appearance have a litte effect to decrease enzyme activity.Chitin oligosaccharides with degrees of polymerization (DP) ranging from 2 to 5 was production by chitinase from Bacillus sp. SW71 with 0,0085 IU/mg chitin had yielded pentamer unit for reaction during 12 hours.

Acknowlegment

The present work was supported by the Cooperation between Research and Technology Ministry Indonesian Republic (Fundamental Research Insentive 2010 and 2011) and Research Center for Biotechnology, Bogor Agricultural University with Haluoleo university, Kendari.

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References

Arnold, L.D., and N.A. Solomon. 1986. Manual of industrial microbiology and biotechnology. Washington: American Society for Microbiology. Bhatnagar, A., and M. Sillanpaa . 2009. Applications of chitin- and chitosan-derivatives for the detoxification of water and wastewater--a short review. Adv Colloid Interface Sci. 152(1-2):26- 38. Curroto, E., And F.Aros . 1993. Quantitative determination of chitosan and the percentage of free amino groups. Anal Biochem. 211: 240-241. Dahiya, N., R. Tewari, G. Hoondal. 2005. Biotechnological aspects of chitinolytic enzymes: a review. Appl Microbiol Biotechnol. 2005. 25:1-10. Flach, J., P.E. Pilet and Jolles. 1992. Whats new in chitinase research?. Experientia. 48:701-716. Hertriyani. 2005. Produksi oligomer kitin dari enzim kitinase dan aktivitasnya terhadap proliferasi sel limfosit manusia secara in vitro. [skripsi]. Fateta. Institut Pertanian Bogor. Ilyina, A.V., N. Yu , Tatarinova, P. Valeri, Varlamov. 2000. The preparation of low-molecular-weight chitosan using chitinolitic complex from Streptomyces kurssanovii. Process. Biochem. 34:875- 878. Imoto, T., and K. Yagashita . 1971. A simple activity measurement of lysozyme. Agric. Biol. Chem. 35:1154-1156. Karita, S., A. Ohtaki, M. Noborikawa, and K. Nakasaki. 2001. A cryptic plasmid , pAO1, from a compost bacterium, Bacillus sp. Biosci. Biotechnol. Biochem. 65(1): 226-228. Kolodziejska, I., P.A. Wojtasz, G. Ogonowska, Z.E. Sikorski. 2000. Deacetylation of chitin in two-stage chemical and enzymatic process. Bulletin of The Sea Fisheries Institute. 2:15-24. Meidina. 2005. Aktivitas anti bakteri oligomer kitosan hasil degradasi oleh kitosanase Bacillus licheniformis MB-2. [tesis]. Sekolah Pascasarjana, Institut Pertanian Bogor. Murao, S., T. Kawada, H. Itoh, H. Oyama, T. Shin. 1992. Purification and characterization of novel type of chitinase from Vibrio alginolyticus TK 22. Bioschi, Biotechnol. Biochem. 56:368-369. Muzarelli, R.A.A. 2010. Chitin and chitosans as immunoadjuvants and non-allergenic drug carriers . Marine Drugs. 8:292-312. Nam-Soo, K. and S. Kim. 1991. Some molecular characteristics and improving methods for thermal stability of enzyme. Kor. J. Appl. Microbiol. Biotechnol. 19(1):100-108. Roby, D., A. Gadelle and A.Toppan. 1987. Biochim. Biophys.. Res. 143: 885-892. Sakai, K., A. Yaota, H. Kurokawa, M. Wakayama and M. Moriguchi. 1998. Prification and characterization of three thermostable endochitinase of a Bacillus noble strain MH-1 isolated from chitin containing compost. Appl. Environ. Microbiol. 64(9):3397-3402. Semenuk, T., P. Krist, J. Pavlicek, K. Bezouska, M. Kuzma, P. Novak, V. Kren. 2001. Syntesis of chitooligomer-based glycoconjugates and their binding to the rat Natural Killer cell activation receptor NKR-P1. Glycoconj. 18:817-26. Suresh, P.V. and M. Chandrasekaran. 1999. Impact of process parameters on chitinase production by an alkalophilic marine Beauveria bassiana in solid state fermentation. Process Biochemistry.34:257-267. Suzuki, S. 1996. Studies of biological effects of water soluble lower homolougous oligosacharide of chitin and chitosan. Fragrance. 15:61-68.

619 | ICSAFS-UNPAD Tanigawa, T., Y. Tanaka, H. Sashiwa, H. Saimoto, Y. Shigemasa. 1991. Effect of Partially Deacetylated chitin Oligomers on Two Macrophage Cell Lines: Comparison with Lipopolysaccharide and Phorbol Myristate Acetate. Yonago Acta medica. 34:155-162. Trombotto, S., C. Ladaviere, F. Delolme, A. Domard. 2008. Chemical preparation and structural characterization of a homogeneous series of chitin/chitosan oligomers. Biomacromolecules.9(7): 1731-1738. Ueda, M. and M. Arai. 1992. Purification and some properties of chitinases from Aeromonas sp. No. 10S-24. Biosci. Biotech. Biochem. 56(3):460-464. Ueda, M., A. Fujiwara, T. Kawaguchi, M. Arai. 1995. Purification and some properties of six chitinase from Aeromonas sp. No. 10S-24. Bioschi Biotechnol Biochem. 60:162-164. Usui, T., H. Matsui and K. Isobe. 1990. Enzymic syntesis of chitooligosaccharides utilizing transglycosylation by chitinolytic enzyms in a buffer containing amonium sulfate. Carbohydrate Research 203: 65-77. Wahyuni, S. 2006. Aktivitas kitooligomer hasil reaksi enzimatik terhadap proliferasi sel limfosit dan sel kanker. [disertasi]. Sekolah Pascasarjana, Institut Pertanian Bogor. Watanabe, T., T. Yamada, W. Oyanagi, K. Suzuki, H. Tanaka. 1992. Purification and some properties of chitinase B1 from Bacillus circulans WL-12. Biosci. Biotech. Biochem 56: 682-683. Wang, S.L., W.T. Chang, M.C. Lu. 1995. Production of chitinase by Pseudomonas aeruginosa using shrimp and crab powdwe as a carbon source. Proc. Natl. Sci. Counc.ROC B. 19:105-112. Wang, S.L., W.T. Chang. 1997. Purification and characterization of two bifunctional chitinase/lysozymes extracellularly produced by Pseudomonas aeruginosa K-187 in a shrimp and crab shell powder medium. Appli. Environt. Microbiol. 63:380-386. Yeon, J.C., E.J. Kim, Z. Piao, Y.C. Yun, Y.C. Shin. 2004. Purification and characterization of chitosanase from Bacillus sp. strain KCTC 0377BP and Its application for the production of chitosan oligosaccharides. Appl. and Environ. Microbiol. 70:4522–4531. Yoon, J.H. 2005. Enzymatic synthesis of chitooligosaccharides in organic cosolvents. Enzyme and Microbial Technology. 37(6):663-668.

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2 Anand Niketan College of Agriculture Warora – 442914. Dist: Chandrapur. Maharashtra. India 3 Padang, Indonesia 4 Bale Bandung University Bandung, Jawa Barat, Indonesia 5 Batanghari University, Jambi Jambi, Indonesia 6 Bogor Agricultural University Dramaga Campus, Bogor 16610, Indonesia 7 Brawijaya University Jalan. Veteran, Malang, 65145, Indonesia 8 College of Agriculture, Purdue University 3 , 610 Purdue Mall, West Lafayette, USA 9 , Tembalang Campus, Semarang 50275, Indonesia 10 Flinders University Adelaide, Australia 11 Gaziosmanpasa University Tokat, Turkey 12 Haluoleo University Kendari, , Indonesia 13 Haluoleo University Kendari 93232, Sulawesi Tenggara, Indonesia. 15 Indonesia University of Education Bandung, Indonesia 16 Institut Teknologi Bandung Jatinangor, Sumedang 45 363, Indonesia 17 Institute for Animal Husbandry, Belgrade Zemun - Serbia

18 Institute of Bio-IT Selangor Uisels Shah Ala Capus, A/A of Ziko Road, Setio 7, 40000 Shah Alam 19 Institute of Economics and Rural Bamako, Mali Development 20 Islamic Azad University Varamin Branch, Iran

21 Islamic Science University of Malaysia, 78000 Nilai, Negeri Sembilan, Malaysia

23 Jambi University Jambi, Indonesia

24 Jember University Jember 68121, Indonesia

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26 Justus Liebig University Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany

27 Kansas State University Manhattan, Kansas, USA

28 Kasetsart University 50 Phahon Yothin Road, Chatuchak, Bangkok, 10900, Thailand 29 Lanzhou University Lanzhou 730000, P.R. China

30 Leibniz Institute Dummerstorf, Germany

31 Nanyang Technological University Singapore

32 Nommensen University Medan, North , Indonesia

33 Pasundan University Bandung, Indonesia

34 Satya Wacana Christian University Jalan Diponegoro 52-60 Salatiga-50711, Indonesia

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40 Tohoku University Sendai, Japan

42 Universitas Al Azhar Indonesia Jakarta, Indonesia

43 Universitas Gadjah Mada Jalan Fauna no. 2, Karangmalang, Yogyakarta 55281, Indonesia 44 Universitas Islam "45" Bekasi 17113,Indonesia

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49 Universitas Pembangunan Nasional Yogyakarta 55283, Indonesia Vetea, Yogakata 50 Universiti Malaysia Terengganu 21030 Kuala Terengganu, Terengganu, Malaysia

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69 Centre for Conservation and Rehabilitation Jalan Gunung Batu 5, Bogor, 16610, Indonesia Research and Development 70 Assesment Institute for Agricultural Kupang, 85001, Indonesia Technology (BPTP) Nusa Tenggara Timur

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71 Assesment Institute for Agricultural Jalan Chr. Soplanit Rumah Tiga, Poka, Ambon, Maluku, Technology (BPTP), Moluccas Indonesia 72 Assessment Institute For Agricultural Kendari, Southeast Sulawesi, Indonesia Technology (BPTP) Kendari

73 Badan Meteorologi, Klimatologi, dan Kupang 85001, Indonesia Geofisika 74 Badan Tenaga Nuklir Nasional Jakarta 12070, Indonesia

75 Center for Agricultural Technology Bogor Indonesia Assessment and Development 76 Centre for Pharmaceutical and Medical Jakarta, Indonesia Technology, Badan Pengkajian dan Penerapan Teknologi 77 East Java Assesment Institute for Jalan Raya Karangploso Km 4 PO Box 188 Malang 65101 Agriculture Technology Indonesia 78 Indonesia Research Institute for Tobacco Jalan Raya Karangploso PO Box 199 Malang, Indonesia and Fiber Crops 79 Indonesian Center for Agricultural Bogor 16114, Indonesia Postharvest Research Development (ICAPRD) 80 Indonesian Legumes and Tuber Crops Jalan Raya Kendalpayak Km 8 Malang 65101- East Java, Research Institute Indonesia.

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