fructose, which can be used to make

fructose, which can be used to make syrup. The sweetness of syrup
is about 20% higher than that of sucrose but the crystallinity of syrup
is low. And the color of syrup is shallower than that of the products
of sucrose by acid hydrolysis. Thus, -d-fructofuranosidase is
widely used in foodstuff and vintage industry [10] and is also
widely used in fermentation industry to produce alcohol [11],
lactic acid, glycerol, and man-made honey [12]. Furthermore,
-d-fructofuranosidase can also be used in cosmetic, medicament,
and paper making. However, the higher cost, lower activity in vitro,
and instability of -d-fructofuranosidase restrict its wider use in
industry. Some investigators immobilized -d-fructofuranosidase
onto silicon layer [13], hydroxyethyl methacrylic acid resin [14],
and nanometer hydrogel [15]. Although these practices can
increase its stability, the activities of the immobilized enzymes are
decreased greatly.
The purpose of this study was to promote the activity and
stability of yeast -d-fructofuranosidase (YFF) through chemical
modification with COS. In this study, COS was used to chemically modify YFF. The effects of pH, temperature, reaction time,
the ratio of COS and enzyme dosage, and substrate concentrations
on enzyme modification were investigated. Furthermore, the variations of the conformation and the configuration of the enzyme
between the modified- and unmodified-YFF were comparatively
analyzed by the methods of ultraviolet absorption spectrum, ultraviolet differential spectrum and fluorescence emission spectrum.
The results indicated that chemical modification of yeast YFF with
appropriate COS molecules significantly improved the activity and
stability of YFF through a relatively cheap and efficient way.
2. Materials and methods
2.1. Material and equipments
Yeast -d-fructofuranosidase (YFF), 2,4,6-trinitrobenzene sulfonic acid (TNBS),
bovine serum albumin (BSA), and sucrose were purchased from Sigma Company
(St. Louis, MO, USA). Coomassie bright blue G-250 and Coomassie bright blue R-250
were purchased from Fluka Company. d-Fructose and d-glucose were purchased
from Amresco Company. Chitooligosaccharide (COS) was offered by Shanghai Taohe
Biological Technical Co (Shanghai, China). All the other chemicals were of analytical
grade.
Gel Imaging System was obtained from Zhuhai Heima Co. (Zhuhai, Guangdong,
China). TFD5503 Freeze-Dryer was purchased from Shin Lab Co. Ltd. (South Korea).
Nicolet AVATAR 360 Fourier Infrared Spectrometer was obtained from E.S.P Co.
(Eatontown, NJ, USA). UV3010 Spectrophotometer and F4500 Fluorescence Spectrometer were purchased from Hitachi Co. (Japan).
2.2. Identification of YFF purity
Polyacrylamide gel electrophoresis (PAGE) was employed to identify the purity
of merchandize YFF. The concentration of spacer gel was 4.5% (w/v) and separation
gel was 7.5% (w/v). The sample applied was 10 L/well. The voltages were initially
set at 150 V when the sample was running in spacer gel and changed to 200 V when
the sample reached the separation gel. After electrophoresis the gel was unglued,
and stained with Coomassie bright blue R-250 solution for 30 min and destained by
0.25 mol/L NaCl for 10 h [16]. The pictures of the gel were taken by using gel-imaging
system.
2.3. Chemical modification of YFF with COS
2.3.1. Selection of appropriate modifiers in different molecular weight
Chitooligosaccharide (COS) with an average MW of 5000 and chitosan with an
average MW of 20,000 were used to modify YFF.
A reaction system containing 25 mg YFF dissolved in 5 mL of 0.1 mol/L pH 4.0
sodium acetate buffer, 5 mL of 0.1 mol/L NaIO4and 0.5 g of sucrose was stirred for
30 min. The reaction was stopped by adding 400 L of glycol. The mixed solution
was then dialyzed against a 2 L of 0.1 mol/L sodium acetate buffer for 4 h. Dialysis
was repeated 3 times.
The activated YFF solution was mixed with 0.5 g of sucrose and 40 mg of COS or
chitosan, which were dissolved in 2 mL of 0.1 mol/L pH 4.0 sodium acetate buffer,
and stirred for 4 h. One mL of 1 mol/L NaBH4solution was then added and stirred for
another 4 h. The reaction mixture was dialyzed in a 2 L of 0.1 mol/L sodium acetate
buffer for 4 h. Dialysis was repeated 3 times. Then the modified enzyme, named
COSFF, was obtained.

Hasil (Bahasa Indonesia) 1:

fruktosa, yang dapat digunakan untuk membuat sirup. Manisnya sirupsekitar 20% lebih tinggi daripada yang sukrosa tetapi bagian kristalinitas siruprendah. Dan warna sirup dangkal daripada produksukrosa hidrolisis asam. Dengan demikian, adalah -d-fructofuranosidasesecara luas digunakan dalam industri pangan dan vintage [10] dan jugabanyak digunakan dalam industri fermentasi untuk menghasilkan alkohol [11],asam laktat, gliserol, dan madu buatan manusia [12]. Selain itu,-d-fructofuranosidase juga dapat digunakan dalam kosmetik, medikamen,dan membuat kertas. Namun, biaya yang lebih tinggi, lebih rendah aktivitas di vitro,dan ketidakstabilan -d-fructofuranosidase membatasi penggunaannya yang luas dalamindustri. Beberapa penyelidik bergerak -d-fructofuranosidaseke silikon lapisan [13], resin asam methacrylic hes [14],dan nanometer hydrogel [15]. Meskipun praktek-praktek ini dapatmeningkatkan stabilitas, aktivitas enzim bergeraksangat menurun.Tujuan dari penelitian ini adalah untuk mempromosikan kegiatan danstabilitas ragi -d-fructofuranosidase (YFF) melalui kimiaModifikasi dengan COS. Dalam studi ini, COS digunakan untuk kimia memodifikasi YFF. Efek dari pH, suhu, waktu reaksi,rasio COS dan dosis enzim, dan konsentrasi substratpada enzim modifikasi diselidiki. Selain itu, variasi konformasi dan konfigurasi enzimantara diubah - dan unmodified-YFF yang relatifdianalisis dengan metode ultraviolet spektrum penyerapan, diferensial spektrum ultraviolet dan spektrum emisi fluoresensi.Hasilnya mengindikasikan modifikasi kimia ragi YFF dengansesuai COS molekul secara signifikan meningkatkan aktivitas danstabilitas YFF melalui cara yang relatif murah dan efisien.2. bahan dan metode2.1. bahan dan peralatanRagi -d-fructofuranosidase (YFF), 2,4,6-trinitrobenzene sulfonat asam (TNBS),sapi albumin serum (BSA), dan sukrosa dibeli dari perusahaan Sigma(St. Louis, MO, USA). G Coomassie-250 biru cerah dan cerah Coomassie biru R-250dibeli dari perusahaan Fluka. d-fruktosa dan d-glukosa yang dibelidari perusahaan Amresco. Chitooligosaccharide (COS) yang ditawarkan oleh Shanghai TaoheBiologis teknis Co (Shanghai, Cina). Semua bahan kimia lain yang analitiskelas.Gel Imaging sistem Diperoleh dari Zhuhai Heima Co (Zhuhai, Guangdong,Cina). TFD5503 beku-Dryer dibeli dari Shin Lab Co Ltd (Korea Selatan).Nicolet AVATAR 360 Fourier spektrometer inframerah Diperoleh dari ESP Co(Eatontown, NJ, USA). UV3010 Spectrophotometer dan F4500 fluoresensi spektrometer yang dibeli dari Hitachi Co (Jepang).2.2. identifikasi YFF kemurnianElektroforesis polyacrylamide gel (halaman) digunakan untuk mengidentifikasi kemurniandari barang YFF. Konsentrasi spacer gel adalah 4,5% (w/v) dan pemisahangel adalah 7,5% (w/v). Sampel yang diterapkan pada 10 L/baik. Tegangan awalnyaTerletak di 150 V ketika sampel ini berjalan di spacer gel dan berubah menjadi 200 V ketikasampel mencapai gel pemisahan. Setelah Elektroforesis gel adalah unglued,diwarnai dengan biru cerah Coomassie solusi R-250 selama 30 menit dan destained oleh0.25 mol/L NaCl untuk 10 h [16]. Gambar-gambar dari gel yang diambil dengan menggunakan pencitraan gelsistem.2.3. kimia modifikasi YFF dengan COS2.3.1. pemilihan sesuai pengubah berat molekul yang berbedaChitooligosaccharide (COS) dengan MW 5000 dan chitosan dengan rata-ratarata-rata MW 20.000 digunakan untuk memodifikasi YFF.Sistem reaksi yang mengandung 25 mg YFF dilarutkan dalam 5 mL 0,1 mol L pH 4.0natrium asetat buffer, 5 mL 0,1 mol/L NaIO4and 0,5 gram sukrosa diaduk selama30 menit. Reaksi dihentikan oleh menambahkan 400 L glikol. Campuran larutankemudian dialyzed terhadap 2 L 0,1 mol/L natrium asetat buffer untuk 4 h. dialisisdiulang 3 kali.Solusi YFF diaktifkan dicampur dengan 0,5 gram Sukrosa dan 40 mg cos atauchitosan, yang telah dilarutkan dalam 2 mL 0,1 mol L pH 4.0 natrium asetat buffer,dan bergerak untuk 4 h. Satu mL 1 mol/l NaBH4solution kemudian ditambahkan dan diaduk selamalain 4 h. Campuran reaksi dialyzed di 2 L 0,1 mol/L natrium asetatpenyangga untuk 4 h. dialisis diulang 3 kali. Kemudian enzim modifikasi, bernamaCOSFF, diperoleh.