Halina Kalinowska

Antarctic marine bacterium Pseudoalteromonas sp. 22b as a source of cold-adapted β-galactosidase

The marine, psychrotolerant, rod-shaped and Gram-negative bacterium 22b (the best of 41 beta-galactosidase producers out of 107 Antarctic strains subjected to screening), classified as Pseudoalteromonas sp. based on 16S rRNA gene sequence, isolated from the alimentary tract of Antarctic krill Thyssanoessa macrura, synthesizes an intracellular cold-adapted beta-galactosidase, which efficiently hydrolyzes lactose at 0-20 degrees C, as indicated by its specific activity of 21-67 U mg(-1) of protein (11-35% of maximum activity) in this temperature range, as well as k(cat) of 157 s(-1), and k(cat)/K(m) of 47.5 mM(-1) s(-1) at 20 degrees C. The maximum enzyme synthesis (lactose as a sufficient inducer) was observed at 6 degrees C, thus below the optimum growth temperature of the bacterium (15 degrees C). The enzyme extracted from cells was purified to homogeneity (25% recovery) by using the fast, three-step procedure, including affinity chromatography on PABTG-Sepharose. The enzyme is a tetramer composed of roughly 115 kDa subunits. It is maximally active at 40 degrees C (190 U mg(-1) of protein) and pH 6.0-8.0. PNPG is its preferred substrate (50% higher activity than against ONPG). The Pseudoalteromonas sp. 22b beta-galactosidase is activated by thiol compounds (70% rise in activity in the presence of 10 mM dithiotreitol), some metal ions (K(+), Na(+), Mn(2+)-40% increase, Mg(2+)-15% enhancement), and markedly inactivated by pCMB and heavy metal ions, particularly Cu(2+). Noteworthy, Ca(2+) ions do not affect the enzyme activity, and the homogeneous protein is stable at 4 degrees C for at least 30 days without any stabilizers.

Purification and characterization of two endo-1,4-β-xylanases from Antarctic krill, Euphausia superba Dana

Two Euphausia superba Dana endo-1.4-beta-xylanases (A, and B), hydrolysing xylan in the same manner as the enzyme classified as EC 3.2.1.8, were isolated and purified. (2) The enzymes were distinguished by their molecular mass and charge, affinities towards the oat xylan (Km of 4.1 and 7.7 mg ml(-1), respectively), values of activation energy in oat xylan hydrolysis (35.5 and 42.5 kJ mol(-1), respectively), as well as the way in which they split the substrate. (3) In vitro they showed the same optimal temperature (37-40 degrees C), optimal pH (5.7-6.0), very low thermostability, and were stabilized and activated by Ca2+ and Mg2+ ions, as well as by some unidentified substances with molecular mass less than 17 kDa, present in crude extracts of krill.

A cold-adapted esterase from psychrotrophic Pseudoalteromas sp. strain 643A

A psychrotrophic bacterium producing a cold-adapted esterase upon growth at low temperatures was isolated from the alimentary tract of Antarctic krill Euphasiasuperba Dana, and classified as Pseudoalteromonas sp. strain 643A. A genomic DNA library of strain 643A was introduced into Escherichiacoli TOP10F’, and screening on tributyrin-containing agar plates led to the isolation of esterase gene. The esterase gene (estA, 621 bp) encoded a protein (EstA) of 207 amino acid residues with molecular mass of 23,036 Da. Analysis of the amino acid sequence of EstA suggests that it is a member of the GDSL-lipolytic enzymes family. The purification and characterization of native EstA esterase were performed. The enzyme displayed 20–50% of maximum activity at 0–20°C. The optimal temperature for EstA was 35°C. EstA was stable between pH 9 and 11.5. The enzyme showed activity for esters of short- to medium-chain (C4 and C10) fatty acids, and exhibited no activity for long-chain fatty acid esters like that of palmitate and stearate. EstA was strongly inhibited by phenylmethylsulfonyl fluoride, 2–mercaptoethanol, dithiothreitol and glutathione. Addition of selected divalent ions e.g. Mg2+, Co2+ and Cu2+ led to the reduction of enzymatic activity and the enzyme was slightly activated (∼30%) by Ca2+ ions.

Collagenolytic serine proteinase from Euphausia superba Dana (Antarctic krill).

1. A serine proteinase isolated from E. superba shows collagenolytic properties: it acts on collagens from Achilles tendon (type I and V) and reconstituted fibrils of calf skin collagen under conditions that do not denature the substrates. 2. At 25 degrees C and pH 7.5 the enzyme both splits the calf skin collagen in solution to the fragments TCA and TCB and catalyses the conversion of dimeric molecules to monomeric chains. 3. The enzyme exhibits strong chymotrypsin-like and lower trypsin-like activities. 4. All the enzyme activities are inhibited to the same degree by diisopropylfluorophosphate (DFP), phenylmethylsulphonyl fluoride (PMSF), N alpha-tosyl-L-lysine chloromethyl ketone (TLCK), soybean trypsin inhibitor (SBTI), chicken ovomucoid (CHOM), chymostatin and leupeptin. None of the activities is inhibited by chelating agents and L-cysteine. 5. pH-Optima of the proteinase in protein substrates hydrolysis (6.0-6.2) are lower than those of synthetic substrates cleavage (7.8-8.0 in the case of BzTyrOEt and 8.7-8.9 for BzArgOEt). 6. Four from nine cysteine residues present in the enzyme molecule possess free thiol-groups. Since the enzyme is inhibited by p-chloromercuribenzoate (pCMB), N-ethylmaleimide (NEM) and iodoacetic acid (IAA), the role of its thiol-groups has been discussed.

Biosynthesis and properties of an extracellular metalloprotease from the Antarctic marine bacterium Sphingomonas paucimobilis

An extracellular protease from the marine bacterium Sphingomonas paucimobilis, strain 116, isolated from the stomach of Antarctic krill, Euphausia superba Dana, was purified and characterized. The excretion of protease was maximal at temperatures from 5 to 10°C, i.e. below the temperature optimum for the strain growth (15°C). The highly purified enzyme was a metalloprotease [sensivity to ethylenediaminetetraacetic acid (EDTA)] and showed maximal activity against proteins at 20–30°C and pH 6.5–7.0, and towards N-benzoyl-tyrosine ethyl ester (BzTyrOEt) at pH 8.0. At 0°C the enzyme retained as much as 47% of maximal activity in hydrolysis of urea denatured haemoglobin (Hb) (at pH 7.0), and at −5 and −10°C, 37 and 30%, respectively. The metalloprotease was stable up to 30°C for 15 min and up to 20°C for 60 min. These results indicate that the proteinase from S. paucimobilis 116 is a cold-adapted enzyme.

A low-cost method for obtaining high-value bio-based propylene glycol from sugar beet pulp

A new low-cost pathway for the production of high-value propylene glycol (PG) is proposed. This route of waste biomass utilization employs catalytic reduction of lactic acid obtained from fermented enzymatic digests of sugar beet pulp.

Application of byproducts from food processing for production of 2,3-butanediol using Bacillus amyloliquefaciens TUL 308

ABSTRACT A nonpathogenic bacterial strain Bacillus amyloliquefaciens TUL 308 synthesized minor 2,3-butanediol (2,3-BD) amounts from glucose, fructose, sucrose, and glycerol, and efficiently produced the diol from molasses and hydrolysates of food processing residues. Batch fermentations yielded 16.53, 10.72, and 5 g/L 2,3-BD from enzymatic hydrolysates of apple pomace, dried sugar beet pulp, and potato pulp (at initial concentrations equivalent to 45, 20, and 30 g/L glucose, respectively), and 25.3 g/L 2,3-BD from molasses (at its initial concentration equivalent to 60 g/L saccharose). Fed-batch fermentations in the molasses-based medium with four feedings with either glucose or sucrose (in doses increasing their concentration by 25 g/L) resulted in around twice higher maximum 2,3-BD concentration (of about 60 and 50 g/L, respectively). The GRAS Bacillus strain is an efficient 2,3-BD producer from food industry byproducts.

Effect of Cellulases and Xylanases on Refining Process and Kraft Pulp Properties

Samples of bleached kraft pine cellulosic pulp, either treated with an enzyme preparation (a Thermomyces lanuginosus xylanase, an Aspergillus sp. cellulase, and a multienzyme preparation NS-22086 containing both these activities) or untreated, were refined in a laboratory PFI mill. The treatment with cellulases contained in the last two preparations significantly improved the pulp’s susceptibility to refining (the target freeness value of 30°SR was achieved in a significantly shorter time), increased water retention value (WRV) and fines contents while the weighted average fiber length was significantly reduced. These changes of pulp parameters caused deterioration of paper strength properties. The treatment with the xylanase, which partially hydrolyzed xylan, small amounts of which are associated with cellulose fibers, only slightly loosened the structure of fibers. These subtle changes positively affected the susceptibility of the pulp to refining (refining energy was significantly reduced) and improved the static strength properties of paper. Thus, the treatment of kraft pulps with xylanases may lead to substantial savings of refining energy without negative effects on paper characteristics.

Conversion of various types of lignocellulosic biomass to fermentable sugars using kraft pulping and enzymatic hydrolysis

The aim of this work was to assess the utility of seven different kraft pulps produced from softwood (pine), hardwood (poplar, birch and beech), wheat straw and hemp (bast and harl) as potential sources of sugar feedstocks for fermentation processes. The pulps contained low amounts of hemicelluloses (1.9–8.2% d.w.) and lignin (1.7–15% d.w.). The crystallinity index values ranged from 55% (wheat straw pulp) to 79% (hemp bast pulp), while the average DP varied from around 230 (hemp bast pulp) to 1482 (poplar and birch pulps). The results of enzymatic hydrolysis showed that not only the residual lignin content but also the cellulose crystallinity index decided on the sugar yields while the average polymerization degree had a weak impact. More reducing sugars were obtained from the hardwood pulps and wheat straw pulp (100% d.w.) than from the pine pulp (around 89% d.w.) and two hemp pulps (40.5% d.w. and 44.7% d.w. from the bast and harl pulps, respectively). Glucose was the dominating (69–79% w/w) soluble sugar in enzymatic hydrolysates of the pulps. The sugar profiles of these hydrolysates make them suitable sugar feedstocks for fermentation processes.

Enzymatic Conversion of Sugar Beet Pulp: A Comparison of Simultaneous Saccharification and Fermentation and Separate Hydrolysis and Fermentation for Lactic Acid Production

This study compares the efficiency of lactic acid production by separate hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF) of sugar beet pulp, a byproduct of industrial sugar production. In experiments, sugar beet pulp was hydrolyzed using five commercial enzymes. A series of shake flask fermentations were conducted using five selected strains of lactic acid bacteria (LAB). The differences in the activities of the enzymes for degrading the principal sugar beet pulp components were reflected in the different yields of total reducing sugars. The highest yields after hydrolysis and the lowest quantities of insoluble residues were obtained using a mixture (1:1) of Viscozyme® and Ultraflo® Max. In the SHF process, only a portion of the soluble sugars released by the enzymes from the sugar beet pulp was assimilated by the LAB strains. In SSF, low enzyme loads led to reduction in the efficiency of sugar accumulation. The risk of carbon catabolic repression was reduced. Our results suggest that SSF has advantages over SHF, including lower processing costs and higher productivity. Lactic acid yield in SSF mode (approx. 30 g/L) was 80-90% higher than that in SHF.